Establishing combinatorial screening platform coupled to single-cell analysis

Division Of Basic Sciences - Nci

Investigators

Abstract

Hybrid guide RNA cassettes have been engineered to harbor capture sequences within Cas9 and Cas12a guide RNAs, which make them amenable to capture using 10x Genomics protocols and reagents during single-cell analysis. Pilot experiments have been performed to assess capture efficiency of hgRNAs along with polyadenylated transcripts. Following those results we have further modified the hgRNA expression cassettes to improve the simultaneous capture of Cas9 and Cas12a guides while retaining efficient editing efficiency of our combinatorial system. The guide design has now been fully optimized and we are in the process of perform genetic screening with single-cell transcriptomic readouts, thereby allowing us to gain mechanistic insights into the cellular rewiring in response to single or combined genetic perturbations.